Konjenital Adrenal Hiperplazi Vakalarında CYP21A2 Geni Kopya Sayısı Değişimlerinin MLPA Tekniği İle İncelenmesi

Year 2020, Volume: 2 Issue: 1, 1 - 10, 01.03.2020

Bakhtiyar Mammadov Aysel Kalaycı Yiğin , Filiz Özdemir Ahmet Özaydın , Mehmet Seven

https://doi.org/10.38175/phnx.690298

Abstract

Konjenital adrenal hiperplazi (KAH), adrenal steroid yapımının bozukluğu ile oluşan ve otozomal resesif kalıtım gösteren ailesel hastalıklar grubundandır. Adrenal kortekste kolesterolden kortizol sentezini sağlayan beş enzimden herhangi birinin eksikliği sonucu oluşmaktadır. KAH’ın karakteristik özelliği, kuşkulu genital yapı ve bozulmuş cinsel gelişimdir. KAH’ın en sık nedeni ise 21-hidroksilaz enzim eksikliğidir. 21-hidroksilaz enzim eksikliğine bağlı KAH’ın, ‘klasik’ ve ‘klasik olmayan’ olarak 2 tipi vardır. Klinik tablodaki farklılıklar 21-hidroksilaz genindeki mutasyonların oluşturduğu yapısal ve fonksiyonel değişikliklere bağlıdır.
İki steroid 21-hidroksilaz geni (CYP21A2 aktif gen, CYP21P psödogen) 6. kromozomun kısa kolu üzerinde bulunur. Aktif genin defektine neden olan mutasyonlar aktif gen ile psödogenin birbirine çok yakın ve oldukça homolog olmalarından kaynaklanmaktadır. DNA dizisinin intergenik rekombinasyonları sonucunda delesyonlar, duplikasyonlar, gen konversiyonları ve nokta mutasyonlar meydana gelir. CYP21A2’nin psödogeninin bulunması nedeniyle 21-hidroksilaz enzim eksikliğine bağlı gelişen KAH’a sadece CYP21A2 genine yönelik dizi analizi çalışmalarıyla doğru tanı konulmasında çoğu kez aksaklıklar yaşanmaktadır.
Çalışmamızda; Cerrahpaşa Tıp Fakültesi Tıbbi Genetik Anabilim Dalı’na CYP21A2 geni mutasyon analizi için yönlendirilen hastalar klinik ve laboratuvar bulgularıyla tekrar değerlendirilmeye alındı ve dizi analizinde saptanması mümkün olmayan kopya sayısı değişikliklerinin belirlenmesi hedeflendi. Böylece; hastaların tanısını kesinleştirerek, tedavilerinin gecikmeden yapılmasının sağlanması ile birlikte CYP21A2 genine yönelik yapılan mutasyon analizlerinde MLPA yönteminin kolay, nispeten ucuz, hızlı ve güvenilir bir yöntem olduğunun gösterilmesi amaçlanmıştır.
Çalışmaya dahil ettiğimiz 28 hastada uygulamış olduğumuz MLPA yöntemiyle, 1 hastada genin büyük kısmını kapsayan homozigot, 2 hastada heterozigot delesyon ve 1 hastada ise heterozigot duplikasyon saptanmıştır.

Keywords

Konjenital adrenal hiperplazi, CYP21A2, CYP21P, MLPA

Supporting Institution

İSTANBUL ÜNİVERSİTESİ-CERRAHPAŞA, BİLİMSEL ARAŞTIRMA PROJE KOORDİNATÖRLÜĞÜ

Project Number

TTU-2016-22410

Investigation of Copy Number Changes in CYP21A2 Gene By Using MLPA Technique in Patients with Congenital Adrenal Hyperplasia

Abstract

Congenital adrenal hyperplasia (CAH) belongs to the group of familial diseases with autosomal recessive inheritance, which is caused by a disorder of adrenal steroid production. It is caused by the deficiency of any of the five enzymes that provide cortisol synthesis from cholesterol in the adrenal cortex. Characteristic of CAH is ambiguous genital structure and impaired sexual development. The most common cause of CAH is 21-hydroxylase enzyme deficiency. The CAH, which is caused by 21-hydroxylase deficiency, has 2 types as 'classic' and 'non-classical'. The differences in the clinical picture depend on the structural and functional changes caused by mutations in the 21-hydroxylase gene.
The two steroid 21-hydroxylase genes (CYP21A2 active gene, CYP21P pseudogene) are located on the short arm of chromosome 6. The mutations that cause defect of the active gene are due to the fact that active and pseudogene are very close and quite homologous. Deletions, duplications, gene conversions and point mutations occur as a result of intergenic recombination of the DNA sequence. Due to the presence of CYP21A2 pseudogene; there are often problems in diagnosing CAH, which develops due to 21-hydroxylase deficiency only with sequence analysis studies for the CYP21A2 gene.
In this study; patients who were directed to Department of Medical Genetics in Cerrahpasa Medical Faculty for CYP21A2 gene mutation analysis were reevaluated with clinical and laboratory findings. It was aimed to determine the number of copy changes that could not be detected in the sequence analysis. Therefore; ensuring the treatment of the patients without delay in their diagnosis; In addition, it is aimed to demonstrate that MLPA method is an easy, relatively cheap, fast and reliable method in mutation analysis for the CYP21A2 gene.
With the MLPA method, 28 patients were included in the study, homozygous deletion covering the majority of the gene in 1 patient, heterozygous deletion in 2 patients and heterozygous duplication in 1 patient were detected.

Keywords

Congenital adrenal hyperplasia, CYP21A2, CYP21P, MLPA

Project Number

TTU-2016-22410

References

• 1. Speiser PW, Azziz R, Baskin LS, Ghizzoni L, Hensle TW, Merke DP, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010 Sep;95(9):4133-60.
• 2. Delle Piane L, Rinaudo PF, Miller WL. 150 years of congenital adrenal hyperplasia: translation and commentary of De Crecchio's classic paper from 1865. Endocrinology. 2015 Apr;156(4):1210-7.
• 3. Wilkins L, Lewis RA, Klein R, Rosemberg E. The suppression of androgen secretion by cortisone in a case of congenital adrenal hyperplasia. Bull Johns Hopkins Hosp. 1950 Apr;86(4):249-52.
• 4. New MI. An update of congenital adrenal hyperplasia. Ann NY Acad Sci. 2004 Dec;1038:14-43.
• 5. New M, Lekarev O, Lin-Su K, Parsa A, Khattab A, Pina C, et al. Congenital Adrenal Hyperplasia. In: De Groot LJ, Beck-Peccoz P, Chrousos G, Dungan K, Grossman A, Hershman JM, et al., editors. Endotext. South Dartmouth (MA) 2000.
• 6. Turcu AF, Auchus RJ. Adrenal steroidogenesis and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 2015 Jun;44(2):275-96.
• 7. Charmandari E, Brook CG, Hindmarsh PC. Classic congenital adrenal hyperplasia and puberty. Eur J Endocrinol. 2004 Nov;151 Suppl 3:U77-82.
• 8. Turcu AF, Auchus RJ. Novel treatment strategies in congenital adrenal hyperplasia. Curr Opin Endocrinol Diabetes Obes. 2016 Jun;23(3):225-32.
• 9. Kim KS, Kim J. Disorders of sex development. Korean J Urol. 2012 Jan;53(1):1-8.
• 10. Hughes IA. Disorders of sex development: a new definition and classification. Best Pract Res Clin Endocrinol Metab. 2008 Feb;22(1):119-34.
• 11. Ahmed SF, Rodie M. Investigation and initial management of ambiguous genitalia. Best Pract Res Clin Endocrinol Metab. 2010 Apr;24(2):197-218.
• 12. Charmandari E, Chrousos G, Merke DP. Classic Congenital Adrenal Hyperplasia. In: Linos D, van Heerden JA, editors. Adrenal Glands: Diagnostic Aspects and Surgical Therapy. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005. p. 101-13.
• 13. Bas F, Kayserili H, Darendeliler F, Uyguner O, Gunoz H, Yuksel Apak M, et al. CYP21A2 gene mutations in congenital adrenal hyperplasia: genotype-phenotype correlation in Turkish children. J Clin Res Pediatr Endocrinol. 2009;1(3):116-28.
• 14. Kandemir N, Yordam N. Congenital adrenal hyperplasia in Turkey: a review of 273 patients. Acta Paediatr. 1997 Jan;86(1):22-5.
• 15. Charmandari E, Brook CG, Hindmarsh PC. Why is management of patients with classical congenital adrenal hyperplasia more difficult at puberty? Arch Dis Child. 2002;86(4):266-9.
• 16. MD WLMMCEF. Adrenal Cortex and its Disorders. In: Sperling M, editor. Pediatric endocrinology. Fourth edition. ed. Philadelphia, PA: Elsevier/Saunders;2014;p.471,532 pages.
• 17. White PC, Werkmeister J, New MI, Dupond B. Steroid 21-Hydroxylase deficiency and the majör histocompatibility complex. Hum Immunol. 1986;15(4):404-15.
• 18. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev. 2000 Jun;21(3):245-91.
• 19. Nimkarn S, Gangishetti PK, Yau M, New MI. 21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, et al., editors. GeneReviews(R). Seattle (WA)2015.
• 20. Tsai LP, Lee HH. Analysis of CYP21A1P and the duplicated CYP21A2 genes. Gene. 2012 Sep 10;506(1):261-2.
• 21. Jang JH1, Jin DK, Kim JH, Tan HK, Kim JW, Lee SY, Ki CS, Park HD. Multiplex ligation-dependent probe amplification assay for diagnosis of congenital adrenal hyperplasia. Ann Clin Lab Sci. 2011;41(1):44-7.
• 22. Concolino P, Mello E, Toscano V, Ameglio F, Zuppi C, Capoluongo E. Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/duplications in congenital adrenal hyperplasia: first technical report. Clin Chim Acta. 2009 Apr;402(1-2):164-70.
• 23. Choi JH, Jin HY, Lee BH, Ko JM, Lee JJ, Kim GH, Jung CW, Lee J, Yoo HW. Clinical phenotype and mutation spectrum of the CYP21A2 gene in patients with steroid 21-hydroxylase deficiency. Exp Clin Endocrinol Diabetes. 2012 Jan;120(1):23-7.
• 24. Miller, W. L. Phenotypic heterogeneity associated with the splicing mutation in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab. 1997 Apr;82(4):1304.